1. Technical Field
The disclosed subject matter relates to an improved self-assembling protein hydrogel, and, more specifically, to a self-assembling protein hydrogel that includes a bio-active protein.
2. Background Art
A hydrogel is a two (or more) component three-dimensional network of polymer chains dispersed in water. Hydrogels are very absorbent, and may contain over 99% water. Common uses for hydrogels include use as scaffolding in tissue engineering, as a sustained-release drug delivery system, in contact lenses, in biocatalytic electrodes, and in wound dressings. Common monomers used in hydrogel formation include polyvinyl alcohol, sodium polyacrylate, and other acrylate polymers and copolymers with an abundance of hydrophilic groups.
Hydrogels have become of greater interest recently because they allow enzymes and other bio-active proteins to be immobilized on a surface through entrapment in, or chemical attachment to, a gel matrix. Prior attempts to produce hydrogels incorporating enzymes have attempted to chemically attach enzymes to synthetic polymers or entrap enzymes within a hydrogel. These approaches require a great deal of processing, and correspondingly high costs, while at the same time result in a final product which typically has a non-homogenous distribution of enzyme throughout the gel.
Protein-based hydrogels have received comparatively more attention, as, although they would be less stable and degrade over time, would also be self-assembling (thereby reducing costs).
For example, U.S. Pat. No. 6,090,911 to Petka et al. (“Petka”), the contents of which are herein incorporated by reference, describes a prior-art protein hydrogel monomer. Referring to FIG. 15, the basic building block of the Petka hydrogel is a tri-block polypeptide including a soluble, randomly coiled domain 1510 flanked by two helical domains 1520. The helices are characterized by a heptad repeat of the form abcdefg where a and d are leucine, or non-polar, residues and e and g are charged (both negative and positive) residues.
Referring to FIG. 16, the angular orientation of the helical residues is shown. The side chains of the non-polar leucines lie in a plane along the length of a helix; the hydrophobic nature of the plane leads to the formation of coiled coils. At concentrations greater than approximately 1 wt % tetrameric coiled coils form and tend to precipitate while the randomly coiled domain remains soluble and without secondary structure. A hydrogel is formed as the coiled coil junctions form a colloidal dispersion, physically separated by water-soluble randomly coiled chains.
Precipitation of the protein hydrogel occurs at pH less than 5 as the charged residues at positions e 1610 and g 1620 protonate creating a second non-polar plane leading to the formation of higher-ordered oligomeric bundles of coiled coils. In addition, at pH below 4 the charged residues of the soluble region also protonate reducing the proteins hydrophilicity, causing the protein to precipitate from solution. The upper pH bound of gel formation occurs at pH 11-12 (depending on temperature) as the secondary helical structure of the heptads is lost.
The strength of the interactions between helices within a coiled coil can be tailored through modification of the primary structure. The charged residues at e and g form inter-helical salt bridges adding stability to the structure. Replacement of these residues with similarly charged (i.e. residues with equal charge as its inter-helical pair) or un-charged residues can disfavor the formation of coiled coils. Alternatively, additional salt bridges can be formed with the introduction of more oppositely charged residues. The temperature dependency of the upper pH bound of gel formation is related to the state of deprotonation of the positively charged residues within the helix as the ionic interactions stabilizing the structure are reduced at increasing pH. The fewer salt bridges formed, the less thermal energy is required to denature the helix. At pH 11 α-helical secondary structure is lost at temperatures above 30° C. Under acidic conditions (pH>6) α-helical secondary structure persist at temperatures greater than 80° C.
Although Petka describes a protein hydrogel, the hydrogel incorporates no functional bio-active proteins. Insertion of a bio-active peptide sequence into the hydrogel of Petka has also been described, but incorporation of functional bio-active proteins has not yet been described. (See Mi, L.; Fischer, S.; Chung, B.; Sundelacruz, S.; Harden, J. L., Self-Assembling Protein Hydrogels with Modular Integrin Binding Domains. Biomacromol. 2006, 7, 38-47). Accordingly, there exists a need for a protein hydrogel incorporating a bio-active protein which retains its functionality after gel formation and a technique for producing the same.